Project Summary/Abstract This proposal addresses mechanisms of chromatin assembly, disassembly, and reorganization by the yeast histone chaperone yFACT during transcript elongation. FACT (facilitates chromatin transcription) is a multi- functional protein complex that plays critical roles in several essential cellular processes, including DNA replication, transcription, and repair in all eukaryotes. The mechanisms used by FACT to maintain or alter the properties of chromatin therefore provide insight into the regulation of several fundamental processes. The yeast S. cerevisiae is used here as a powerful model system that allows the combined use of genetic, biochemical, molecular, and single-particle approaches for analysis of the mechanisms of yFACT action that are relevant for all eukaryotes from yeasts to humans. Previous studies performed by our individual groups have resulted in the development of defined, structurally tractable experimental models that recapitulate the important aspects of yFACT action: the histone chaperone activity, and both spontaneous and transcription-dependent reorganization of chromatin. Crucially, while our previous studies have provided a framework for understanding how yFACT affects isolated populations of nucleosomes in vitro and how it alters transcription initiation by affecting chromatin in promoters, the mechanism through which FACT promotes transcription elongation on chromatin templates (the function it is most closely associated with and from which its name is derived) remains poorly understood. We have therefore initiated collaborative studies that take advantage of our distinct, but complementary, individual strengths to examine the effects of yFACT on transcription elongation. Given the initial success of these collaborative efforts (described below), we now propose to extend these studies to examine yFACT in vitro and in vivo, as outlined in this application. We propose to address four primary questions: (1) Which functional domains of FACT and which histone residues mediate yFACT-dependent nucleosome reorganization? (2) How do FACT-histone interactions affect the structure and stability of transcript elongation intermediates? (3) Are these activities mechanistically related to one another? (4) How do FACT's activities contribute to transcript elongation in vivo? These questions will be addressed in vitro using highly purified proteins with homogeneous and well-defined mono- and polynucleosomal chromatin templates by a combination of biochemical, molecular genetic, time-resolved and single-molecule techniques. The effects of yFACT mutations in vitro will be compared with their effects in vivo to gain insight into the physiologically relevant functions of yFACT. Integrating our efforts will allow us to examine the currently mysterious processes that occur when RNA Pol II encounters a nucleosomal barrier during transcription elongation, and how the highly conserved factor yFACT contributes to the goals of both removing and rebuilding this chromatin barrier.